obituary Christian B. Anfinsen The journey not the awiva! matters - Leonard Woolf, after Montaigne CHRISTIAN BOEHMER ANFINSEN died suddenly on May 14,1995 at the age of 79. At the time he was Pro- fessor of Biophysical Chemistry at the Johns Hopkins University, a po- sition he had assumed after his re- tirement from the National Insti- tutes of Health (NIH) in 1981. In 1972 he shared, with Stanford Moore and William H. Stein of Rockefeller University, the Nobel Prize for Chemistry. He had been cited by the Swedish Royal Academy of Sciences for his "studies on ribo- nuclease, in particular the relation- ship between the amino acid se- quence and the biologically active conformation." Chris, as he was known to even the most junior member of his laboratory, was the son of Norwe- gian immigrants from Bergen who had settled in western Pennsylvania. His father, a road construction en- gineer, later moved the family to the Philadelphia area where Chris gradu- ated from Swarthmore College in I937 and went on to study organic chemistry at the University of Penn- sylvania. A fellowship from the American-Scandinavian Foundation sent him to the Carlsberg Labora- tory in Copenhagen in 1939 to study enzyme-based micromethods. The outbreakofwar forced his return to the United States in 1940, but not before he had the chance to see and understand the horrors then gripping Europe. His unusually deep and active sense of so- cial responsibility certainly dated from that period, if not earlier. Chris entered the renowned graduate program in Biological Chemistry at Harvard Medical School where he worked with A. Baird Hastings on a problem in retinal histochemistry. He was awarded his Ph.D. degree in 1943; by 1944 he was working at Harvard in the malaria research project of Vannevar Bush's Office of Scientific Research and Development. Thirty years later some of the observations Chris made then were used in developing the current methods of culturing ma- laria parasites. structural biology volume 2 number 8 august 1995 1916-1995 He returned to the Harvard Bio- logical Chemistry Department in 1946. During the next decade he worked on a variety of projects in what historically may now be seen as the transition phase of biochemistry from the study of intermediary me- tabolism to that of molecular and structural biology. He used mi- cromethods (`Cartesian Divers') for measuring metabolic processes and, with A.K. Solomon, pioneered the use of stable and radioactive isotopes for tracing metabolic pathways, in- cluding the biosynthesis and degrada- tion of proteins. A year (1947-1948) at the Medical Nobel Institute in Stockholm, in Hugo Theorell's labo- ratory, led to partial purification of aconitase, with Jack Buchanan, and further hints of his evolving interest in proteins. In 1950, to the astonishment of his Boston colleagues, Chris did the un- thinkable and gave up his position as Associate Professor at Harvard and moved to the pastures of Bethesda, Maryland to become Chief of the Laboratory of Cellular Physiology in the newly created National Heart In- stitute of the NIH. James Shannon had recently gone to Bethesda as sci- entific director of the new institute and was vigorously recruiting scien- tists to staff a partially renovated Build- ing 3, built in 1938 largely as an ani- mal facility when NIH moved from Washington, DC. Among those who responded to Shannon's vision were Julius Axelrod, Robert Berliner, Rob- ert Bowman, Bernard Brodie, Donald Fredrickson, Edward Korn, Earl and Thressa Stadtman, Daniel Steinberg, and Sidney Udenfriend. They joined Leon Heppel, Bernard Horecker, Herman Kalckar, Arthur Kornberg and others on the staff of the Experi- mental Biology and Medicine Insti- tute. This ensemble of individuals, crowded into the very small building (but later dispersed on the NIH cam- pus after the Clinical Center opened in 1953), was a crucial seed for the explosive growth of biomedical re- search at the NIH that followed. During the next five years, a whole range of publications on plasma lipo- protein metabolism emerged from Chris' lab, as well as a continuing stream of papers on protein structure. While this lipoprotein work was un- doubtedly related to the interests of his new employer, it was never fondly re- membered by him, although the ap- proaches he and his colleagues devel- oped were very important in the sub- sequent clarification of the genetic bases of the lipoprotein diseases. However, it was Fred Sanger's contemporaneous work on the amino acid sequence of insulin that really excited Chris and provided the theme for the remainder of his career. Surely the technique could be made to work to determine the primary structure of an enzyme and perhaps ultimately to synthesize the protein. The availability of a ready supply of bovine pancreatic ribonuclease (Rh'ase) from the Armour Com- pany, a by-product of its protein fractionation work, defined rhe eti- zyme of choice. In 1954, the first paper appeared from Chris's labo- ratory on the structure, cross-link- ages and terminal sequences of RNase. Despite its apparent irrel- evance to heart disease, this work 621 obituary was the beginning of fifteen years of concentrated effort on RNase. In 1954, Chris returned to the Carlsberg Laboratory on a one-year Rockefeller fellowship, to work with Kai Linderstram-Lang, using physical chemistry to study RNase. He joined a remarkable group of scientists, in- cluding Aase Hvidt, Martin Ottesen, Bill Harrington and John Schellman who, over the next decades, contrib- uted greatly to defining the properties of globular proteins. Although, ironi- cally, the main conclusion of the com- bined efforts of this group on RNase that"the data further support the pos- sibility that a considerable part of the enzyme structure may be superfluous from the catalytic standpoint"did not stand up to later work, the structural concepts that Chris added to his chemical and enzymological back- ground were of major importance in the next few years. Upon returning to the NIH it be- came apparent to him that the Moore and Stein team at Rockefeller would likely complete the amino acid se- quence of RNase first. Instead of an all-out effort to compete on the se- quence, he chose to focus on the dif- ficult problem of defining the disulphide cross-links among the eight cysteine residues in RNase. In 1956, during work to develop revers- ible cleavage techniques for the four cystine cross-links, he noted serendipitously that the disulphide bonds could reform with restoration of enzymatic activity, under certain conditions. It was at this moment that Chris'genius for getting to the es- sence of research problems fully mani- fested itself. He quickly realized that the unexpected reversibility of this process, which later included the demonstration of the restoration of secondary and tertiary structure as well as the disulphide bonds, im- plied that the information for the folding of a protein is contained in the amino-acid sequence. The re- sult was also of interest to him in that it implied that the synthesis of func- tional proteins would only require the sequential polymerization of the ap- propriate amino acids. The efforts over the next six years of Chris and his colleagues-especially Fred White, Michael Sela, Ed Haber, Charles Epstein and Robert Goldberger-supplied the detailed ex- perimental analyses of the refolding of in recent years work on chaperones RNase and other proteins to lead to has been interpreted by some to con- the full `thermodynamic hypothesis! tradict Chris' hypothesis. This con- This hypothesis stated that `the three- dimensional structure of a native pro- tein in its normal physiological milieu (solvent, pH, ionic strength, presence of other components such as metal ions or prosthetic groups, tempera- ture, and others) is the one in which the Gibbs free energy of the whole sys- tem is lowest; that is, that the native conformation is determined by the to- tality of interatomic interactions and hence by the amino acid sequence, in a given environment"`. This simple and elegant principle has, over the last third-of-a-century, fusion between.ihermodynamic and kinetic analyses, common in many other areas of science, tnisses the fun- damental nature of the contribution. In fact, Chris and his colleagues- Franc0 delorenzo, David Givol and Sara Fuchs-were the first to identify and characterize a chaperone, the pro- tein-disulphide isomerase. Although Chris participated actively in both ki- netic and equilibrium experiments (including with this author), at heart he favoured a thermodynamic ap- proach. He was more interested in findine out what existed in nature or become part of the fundamental par;- " what one could synthesize, then the digm of molecular biology, as well as details of the process being studied. the basis ofvast biotechnology efforts. From 1962 to 1963, Chris returned The enormous skepticism at the time for a year to Harvard as Professor of among the scientific establishment to Biological Chemistry, but was re- both the experimental results and cruited back to the NIH in 1963 by their interpretation has now been J.E. Rall to head a newly created largely forgotten. Indeed, to some the Laboratory of Chemical Biology in concepts were considered-consistent the National Institute of Arthritis with J.B.S. Haldane's quip about the and Metabolic Diseases. At this response to all new ideas-to have point a new model protein, staphv- been self-evident. On the other hand, lococcal nuclease, was added to h'is Chris photographed in hrs office at the Natlonal Institutes of Health rn the late 1970s with the typewriter (background) that he used to write all his notes and letters 622 structural biology volume 2 number 8 august 1995 obituary Linderstwm-Lang's cancature poster of Chris Anfinsen exhIbIted at the Carlsbera Lab- oratory Christmas party, 1954 (Cour- tesy of Carlsberg Foundation Picture ArchIves, Copen- hagen) experimental program and over the next decade it became the centre of his research interest. Its lack of disulphide bonds and its great stabil- ity made it a better experimental sys- tem than RNase to study the process of protein folding by synthetically al- tering individual amino-acid resi- dues. During this period Chris, and a very large number of young col- leagues, used organic synthetic, chemical, enzymological, biophysi- cal, immunological and genetic ap- proaches to study the properties of this protein and its folding. A fold- ing mechanism incorporating`flick- ering' secondary structure nucle- ation sites and subsequent conden- sation of tertiary structure was evolved that is not much different from today's concepts. By the mid- 1970's Chris' group, with the help of a crystal structure determination by EA. Cotton and his associates, had added `staph' nuclease to the small number of globular proteins, in- cluding hemoglobin, myoglobin, lysozyme as well as RNase, which had been most thoroughly charac- terized. The goal of synthesis of nu- clease and its analogues was partially achieved with `semi-synthesis', by condensation of fragments made by the solid-phase method. Recombi- nant DNA technologies ultimately made this goal fully practical. Along this reseach path Chris- with Iku Kate, Meir Wilchek and Pedro Cuatrecasas--w:< also key in es- tablishing affinity chromatography as a major tool for biochemistry. In the years following these studies and the award of the Nobel Prize, Chris con- tributed significantly to the pu- rification of human interferon and later to the study of highly thermo- stable enzymes. Indeed at the time of his death he was funded by the Na- tional Science Foundation for devel- oping thermostable enzymes for the remediation of environmental con- tamination. This summary of Chris' scientific accomplishments would be incom- plete without mention of several other aspects of his career. In I959 he pub- lished the monograph The A4oleculnr Basis of Evolution'. Appearing at the time of the Darwin centennial, this book was important in initiating the current era of the use of macromo- lecular structure information in evo- lutionary analyses. It was highly in- fluential m shaping the perspective of many of us who entered science in the following decade. His editorship, since 1957, of Advances in Protein Chemistry, most recently with John Edsall, Fred Richards, and David Eisenberg, is considered by many to have been equally influential in defin- ing the field of protein chemistry, Chris was also instrumental in making the NIH much more like a university than a typical government laboratory. He helped create The Foundation for Advanced Education in the Sciences at the NIH and other teaching programs-modelled after the Swarthmore honors seminars- which made the NIH an unusually excellent learning experience for the many young scientists, especially phy- sicians, who came during his three decades there. Chris' laboratory was an interna- tional mecca for scientists. As he said, the best way to promote scientific ex- change among countries is to ex- change scientists. Since his 1957 visit to Israel, at the invitation of Michael Sela and Ephraim and Aaron Katchalski, Chris maintained a deep interest in that country and its science. He frequently visited and served for almost four decades on the Board of Governors of the Weizmann Institute of Science in Rehovot. This deep in- terest later extended to the Hebrew language and the Jewish religion. Chris' sensitivity to political issues was manifest, among other activities, in his work leading up to the 1963 treaty banning atmospheric nuclear testing, in his opposition to the US involvement in Vietnam, and in his human rights activism, especially with regard to scientists in the USSR and in Latin America. For a number of years he chaired the human rights committee of the US National Acad- emy of Sciences. Playing the piano or the viola and, most of all, sailing provided him with limited escapes from science. In sail- ing, as in all things, he was always the optimist; his casualness in his sailboat made these voyages only for the in- trepid. However, when crises occurred on the water, everyone knew that Chris would solve the problem, and he al- ways did. But above all of his professional ac- tivities was Chris' impeccable stan- dards of personal behaviour, whether he was your teacher, colleague, col- laborator, competitor, boss or friend. The roles melded completely into each other; all individuals, from the stock clerk to the director of the Institutes, were treated as fellow crew members in the quest to understand nature. Chris never looked back. Even the Nobel lecture had to have new, unpublished data. Only death has I`- nally stopped a research journey which began with his trip to Copenhagen fifty-six years ago. Alan N. Schechter Laboratory of Chemical Biology, NIDDK, National Institutes of Health, Bethesda, Maryland, USA 20892. Christian Boehmer Anfinsen, bio- chemist; born Monessen, Pennsylva- nia, 26 March 1916; B.A., Swarthmore College 1937; M.S., University of Pennsylvania, 1939; Ph.D., Harvard University Medical School; professor, Harvard 1946-1930,1962-1963; labo- ratory chief, National Institutes of Health, 1950-1962,1963-1981; profes- sor, Weizmann Institute 198 1 - 1982; professor, Johns Hopkins, 1982-1995; Nobel Prize in Chemistry, 1972; mar- riage to Florence Kenenger, 1941.1978 (one son, two daughters); married Libby Shulman Ely 1979; died Pikesville, Maryland, I4 May 1995. 1. Anflnsen. C B Principles that govern the folding of protein chains (Nobel Lecture) Soence 181, 223-230 (1973) 2. Anflnsen, C B The Molecular Bms of Evolution (John Wiley, New York, 1959) structural biology volume 2 number 8 august 1995 623